This invention relates to the production of recombinant polypeptides, and in particular, to the production of synthetic proteins having plural fused domains. More specifically, this invention relates to methods of producing recombinant fusion proteins having dual biological activities such as binding molecules and receptor proteins with preselected specificity and activity. Such fusion proteins are useful, for example, in imaging procedures, in the diagnosis and treatment of various human cancers, infectious diseases, and dysfunctions, and in the development of vaccines.
Fusion proteins having dual activities and/or functions are known and include combinations of peptide hormones, enzymes, transport and receptor proteins, viral coat proteins, interleukins, lymphokines, immunoglobulins (Igs), and fragments thereof. Some of these fusion proteins are known to provide enhanced antigenicity, and therefore, are useful in the production of vaccines. Others have high biological activity, and hence, are useful in treating various infectious and deficiency diseases and disorders. Still other fusion proteins are useful as diagnostic agents because of their enhanced targeting abilities. (See, e.g., U.S. Pat. Nos. 4,223,270, 4,801,536, CA 1217156A, WO 8806630A, and JP 63267278A for examples of such fusion proteins.)
Immunoglobulin molecules have been produced as fusion proteins (e.g., chimeric antibodies). Their structure is particularly conducive to the formation of fused polypeptides having a first protein domain (e.g., a variable region) from a first Ig molecule from one species having a particular specificity, and a second domain (e.g., a constant region) from a second Ig of a different species (and perhaps specificity). They were developed as an alternative to non-chimeric monoclonal antibodies, many of which are of non-human (e.g., murine) origin, and hence may be antigenic to humans. Human monoclonal antibodies are the most desirable therapeutic agents, as their use should provoke a greatly reduced immune response in humans. However, the production of human monoclonal antibodies by cell fusion methodologies is difficult, as immunized human spleen cells are not readily available, and as human hybridomas are notably unstable.
Chimeric antibodies composed of both human and non-human amino acid sequences should elicit less of an immune response in humans, and therefore should have improved therapeutic value. Accordingly, hybrid antibody molecules have been proposed which consist of Ig light (L) and heavy (H) chain amino acid sequences from different mammalian sources. The chimeric antibodies designed thus far comprise variable (V) regions from one mammalian source (usually murine), and constant (C) regions from human or another mammalian source (see, for example, EPO application nos. 84302368.0 (Genentech), 85102665.8 (Research Development Corporation of Japan), and 85305604.2 (Stanford University); P.C.T. application no. PCT/GB85/00392 (Celltech Limited); Morrison et al. (1984) Proc. Natl. Acad. Sci. U.S.A. 81:5851-6855; Boulianne et al. (1984) Nature 312:643-646; and Sahagan et al. (1986) J. Immunol. 137:1066-1074).
The production of recombinant chimeric antibodies with predetermined specificity has typically involved the use of cloned genomic DNA fragments. For example, the genomic DNA sequences encoding H and L chains can be cloned in their rearranged forms (i.e., in the DNA sequence that results from recombination events during B Cell maturation). As such, these genomic sequences contain the information necessary for their expression, (i.e. the 5' untranslated sequences, promoter, enhancer, protein coding region, and donor splice site). The donor splice signals at the 3' end of the V gene segments are compatible with the splice acceptor signals at the 5' end of the Ig regions of other species. That is, the splice product between the two maintains the correct reading frame. For example, when a murine V and a human C.sub.k segment are joined and transfected into the appropriate host cell type, the primary transcript is correctly spliced and results in a mature messenger RNA (mRNA) molecule with an open reading frame through both the V and C regions.
There are disadvantages to the use of genomic V region fragments for the expression of recombinant chimeric antibodies. The first involves the cloning process itself which can be quite laborious for single-copy genes, requiring the screening of many independent clones of a phage library. Furthermore, many hybridomas contain multiple rearranged V genes which represent non-productive recombinational events. The identification of the expressed V.sub.L or V.sub.H segment can often require extensive DNA sequence analysis as well as confirmation by cloning and sequencing the DNA copy of the expressed mRNA (complementary or cDNA).
A more direct approach is to clone the cDNAs for both the L and H chains, and to use cDNA expression vectors for their expression. In this case, cloning is simple and rapid, since Ig mRNA is very abundant in hybridoma cells, and highly efficient methods for cDNA cloning are available. However, when one uses this approach, a problem arises when the separate expression of different V and C regions from different Ig cDNAs is desired, as in the case of chimeric antibodies having, for example, murine V regions and human C regions. The Ig cDNA represents a direct copy of the mRNA which, in turn, is a fusion of V and C exons through normal, in vivo RNA splicing into a continuous polynucleotide sequence. Precise excision and recombination of a murine V.sub.H with a human C.gamma..sub.1, for example, is not possible because appropriate restriction sites are not present at the VC junction of both sequences.
The expression of chimeric antibodies has been accomplished through the use of cloned cDNAs. This procedure may involve the mutagenesis of sequences in both the murine V region and human C region, near the VC junction, such that a common restriction site is created for directly joining the eDNA segments (Liu et al. (1987) Gene 54:33-40).
It is an object of this invention to provide a method of producing fused polypeptides. Another object is to provide methods for producing fusion proteins having dual activities and/or functions such as, for example, chimeric immunoglobulin molecules having a predetermined antigen specificity. Another object is to provide a relatively simple and rapid procedure for providing human/non-human mammalian chimetic antibodies and truncated versions thereof having reduced antigenicity in the human body. Yet another object of the invention is to provide a rapid method of producing a made-to-order chimetic immunoglobulin molecules utilizing a specifically engineered V region gene that can be attached easily to another gene, and transfected and expressed in a host cell.